The present invention relates to a purified form of streptogramins comprising at least one component of the streptogramin B group defined below by the general formula (I) co-crystallized with at least one xe2x80x9cminorxe2x80x9d component of the A group defined below by the general formula (II).
Among the known streptogramins, pristinamycin (RP 7293), an antibacterial product of natural origin produced by Streptomyces pristinaespiralis was isolated for the first time in 1955. The pristinamycin marketed under the name of Pyostacin(copyright) consists mainly of pristinamycin IA and pristinamycin IIA.
Another antibacterial agent of the streptogramin class: virginiamycin, was prepared from Streptomyces virginiae, ATCC 13161 [Antibiotics and Chemotherapy, 5, 632 (1955)]. Virginiamycin (Staphylomycin(copyright)) consists mainly of factor S and factor M1.
U.S. Pat. No. 3,325,359 describes pharmaceutical compositions comprising antibiotic substances constituting the antibiotic 899: factor S and factor M1.
Antibacterial agents of natural origin, of the streptogramin class, consist of a mixture of 2 groups of components: components of the B group and components of the A group, each group having its own particular antibacterial activity. It has been demonstrated that the combination formed by the 2 groups of components gives rise to a synergism of action which results in increased bacteriostatic and bactericidal activity and in broadening of the spectrum of activity.
Components of the streptogramin A and B groups have been described in Streptogramine als Modelsysteme fxc3xcr den Kationentransport durch Membranen, Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Facultxc3xa4t der Georg-August Universitxc3xa4t zu Gxc3x6ttingen, Gxc3x6ttingen 1979, in Antibiotics III, 521 (1975) and in Antibiotics of the virginiamycin family, Inhibitors which contain synergistic components, C. Cocito, Microbiological Reviews, 145-98 (1979). J. Preud""Homme, P. Tarridec, and A. Belloc, Bull. Soc. Chim. Fr., 2, 585 (1968) have also described natural pristinamycin as well as the various components constituting it.
As regards the industrial preparation of products of this class, the techniques available hitherto did not make it possible to obtain, on a preparative scale, a sufficiently purified form and the production of batches of sufficiently constant and reproducible quality to meet the requirements of the registration legislations in certain countries.
In the field of antibacterial agents, it is well known by practitioners that allergies or resistances may develop after administration of certain classes of antibiotics [The New England Journal of Medicine, 324 (9), 601 (1991)]. In hospitals, many resistant strains of Staphylococcus aureus are known in particular. Accordingly, it is extremely useful for doctors to have at their disposal a wide range of chemically different classes so as to be able to adapt the treatment to the specific case of the patient to be treated. The consequence of the absence of commercialization of a given class may be very serious, or even dramatic, since the result can be that patients who do not tolerate the other classes of antibiotics are deprived of treatment.
Thus, the purification tests used always had the aim of removing the minor components from streptogramins, these components being considered as not indispensable and rather as impurities.
Among the components of the A group of natural streptogramins, pristinamycin IIB (PIIB) is a minor component whose weight proportion is less than 10% in natural pristinamycin, and usually about 8% or even about 6% in virginiamycin.
It has now been found, and this forms the subject of the present invention, that the combination consisting of one or more components of the B group, of general formula: 
in which
the symbol R1 represents a methyl or ethyl radical,
the symbol R2 represents a hydrogen atom or a hydroxyl radical, and
the symbol R3 represents a substituted benzyl radical of general formula: 
xe2x80x83in which
1) on the condition that R2 is simultaneously hydrogen, R represents NR4R5 for which one of the symbols R4 and R5 is a hydrogen atom or a methyl radical and the other is a methyl radical and Rxe2x80x2 is a chlorine or bromine atom, or represents an alkenyl radical containing 3 to 5 carbon atoms if R4 and R5 are methyl radicals, or alternatively
2) R is a hydrogen atom and Rxe2x80x2 represents a halogen atom, an alkylamino or dialkylamino radical, an ether oxide residue, an alkylthio radical, an alkyl radical containing 1 to 3 carbon atoms in a straight or branched chain or a trihalomethyl radical, or alternatively
R is a halogen atom, an alkylamino radical containing 2 to 4 carbon atoms in a straight or branched chain, a dialkylamino radical in which the alkyl parts are identical or different and contain 2 to 4 carbon atoms in a straight or branched chain or a methylethylamino radical, a pyrrolidino radical, an alkenylalkylamino radical in which the alkenyl part contains 3 or 4 carbon atoms and the alkyl part is defined as above, a dialkylamino radical in which the alkenyl parts are defined as above, an alkylcycloalkylmethylamino radical in which the alkyl part is defined as above and the cycloalkyl part contains 3 or 4 carbon atoms, an ether oxide residue, an alkylthio radical, an alkylthiomethyl radical, an alkyl radical containing 1 to 6 carbon atoms in a straight or branched chain, an aryl radical or a trihalomethyl radical, and Rxe2x80x2 is a hydrogen atom, or alternatively
R is a halogen atom, an amino, alkylamino or dialkylamino radical, an ether oxide residue, an alkylthio radical, an alkyl radical containing 1 to 6 carbon atoms in a straight or branched chain or a trihalomethyl radical and Rxe2x80x2 represents a halogen atom, an alkylamino or dialkylamino radical, an ether oxide residue or an alkylthio or alkyl radical containing 1 to 3 carbon atoms in a straight or branched chain, and one or more xe2x80x9cminorxe2x80x9d components of the streptogramin A group, of general formula: 
xe2x80x83in which Rxe2x80x3 is a hydrogen atom or a methyl or ethyl radical, is particularly advantageous on account of its in vivo biological activity.
In the general formula (I), when R or Rxe2x80x2 (in R3) represents a halogen atom, it is preferably a fluorine atom, except for if Rxe2x80x2 is a hydrogen atom, in which case R is either chlorine, bromine, fluorine or iodine; when Rxe2x80x2 represents an alkylamino or dialkylamino radical, the alkyl radicals are preferably chosen from methyl and ethyl; when R or Rxe2x80x2 represents is an ether oxide residue, it may be represented by a residue ORxc2x0 for which Rxc2x0 is preferably chosen from a methyl group, an ethyl group optionally substituted with a chlorine atom, an allyl group or a trifluoromethyl group; when R or Rxe2x80x2 represents an alkylthio radical, it is preferably a methylthio radical; when R represents an alkyl radical containing 1 to 6 carbon atoms, it preferably represents methyl, isopropyl or tert-butyl; when R represents an aryl radical, it is preferably a phenyl radical; when R or Rxe2x80x2 represents a trihalomethyl radical, it is preferably a trifluoromethyl radical. Moreover, it is understood that, except where especially mentioned, the alkyl radicals are straight or branched and contain 1 to 4 carbon atoms.
The product of general formula (II) for which Rxe2x80x3 is an ethyl radical, referred to hereinbelow as pristinamycin IIF (PIIF), and the product of general formula (II) for which Rxe2x80x3 is a hydrogen atom, referred to hereinbelow as pristinamycin IIG (PIIG), are products which constitute very minor components of streptogramins, the weight proportion of which components is less than 0.5% in batches of natural product.
The co-crystallization is carried out at a constant stoichiometry of 1 mol of component(s) of general formula (I) with 2 mol of component(s) of the A group of general formula (II) [this stoichiometry corresponding to a relative proportion of about 43-49/57-51 by weight].
According to the invention, the co-crystallized combinations are prepared in the following way: a component of the B group defined by the general formula (I) is added to a crude mixture containing at least 30% of a minor component of the A group corresponding to the general formula (II) dissolved in an organic solvent such as a ketone (acetone, methyl ethyl ketone or methyl isobutyl ketone for example), an ester (ethyl acetate, isopropyl acetate, butyl acetate or isobutyl acetate for example), a chlorinated solvent (methylene chloride, chloroform or 1,2-dichloroethane for example) or a nitrile (acetonitrile for example) to give a co-crystallized compound in the proportions defined above. It is understood that the amount of compound of general formula (I) introduced is conveniently chosen so that the residual concentration of this product (after the co-crystallization) is less than its solubility in the medium. It is also understood that variations in the respective contents of product of general formula (II) and of product of general formula (I) in the initial medium do not lead to any change in the co-crystallized compound obtained.
This co-crystallized combination has the advantage of very good stability and high purity.
The components of the B group of the streptogramins defined by the general formula (I) may be prepared in the following way:
When R3 represents a radical of general formula (III) for which R and Rxe2x80x2 are defined as. in 1), if Rxe2x80x2 is a chlorine or bromine atom, the products of general formula (I) may be obtained by the action of the corresponding N-halosuccinimide derivative on pristinamycin I for which Rxe2x80x2 is a hydrogen atom.
The reaction is carried out using N-chloro- or N-bromosuccinimide in an organic solvent such as, for example, a chlorinated solvent (dichloromethane, dichloroethane or chloroform) or a nitrile (acetonitrile), at a temperature of between 20 and 80xc2x0 C.
When R3 represents a radical of general formula (III) for which R and Rxe2x80x2 are defined as in 1), if Rxe2x80x2 is an alkenyl radical containing 3 to 5 carbon atoms, the products of general formula (I) may be obtained by rearrangement, in a slightly basic medium, of a salt derived from 4-N-alkenylammoniopristinamycin IA of general formula: 
in which R1 is defined as above and R6, R7, R8 and R9 are a hydrogen atom or a methyl radical, provided that at least 2 of them are hydrogen atoms and Xxe2x88x92 represents an anion, to give the derivative of general formula: 
for which R1, R6, R7, R8 and R9 are defined as above.
The reaction is carried out by heating at a temperature of between 80 and 100xc2x0 C. in an aqueous or two-phase medium (for example in ethyl acetate/water medium), in the presence of sodium acetate or sodium or potassium bicarbonate. A 4-N-alkenylammoniopristinamycin IA halide is advantageously used.
The 4-N-alkenylammoniopristinamycin IA halide may be obtained by the action of an alkenyl halide of general formula: 
for which R6, R7, R8 and R9 are defined as above and Hal represents a halogen atom, on a pristinamycin derivative of general formula: 
in which R1 is defined as above.
The reaction is advantageously carried out in an organic solvent such as a chlorinated solvent (for example dichloromethane, dichloroethane or chloroform) or an alcohol (for example ethanol) or in a mixture, at a temperature of between 20xc2x0 C. and the reflux temperature of the reaction mixture. Preferably, a product of general formula (VI) for which Hal is a chlorine or bromine atom is reacted.
When R3 represents a radical of general formula (III) for which R and Rxe2x80x2 are defined as in 2), the products of general formula (I) may be obtained as described below in the examples, from a strain of a streptogramin-producing microorganism possessing at least one genetic modification which affects the biosynthesis of a precursor of the group B streptogramins, the said mutant strain cultured in a suitable culture medium, complemented with at least one original precursor other than that whose biosynthesis is adversely affected (and corresponding to the desired structure), produces the expected streptogramin derivatives.
The strains used in the context of the present invention are thus mutated strains producing natural streptogramins (pristinamycin IA, pristinamycin IB and virginiamycin S). The said genetic modification(s) may be localized either onto one of the genes involved in the biosynthesis of the said precursors or outside of the coding region, for example in regions responsible for the transcriptional or post-transcriptional regulation and/or expression of the said genes or in a region belonging to the transcript containing the said genes.
In particular, the mutant strains possess one or more genetic modifications in at least one of their genes involved in the biosynthesis of precursors of the group B streptogramins. This or these genetic modifications adversely affect the expression of the said gene, that is to say make this gene and, where appropriate, another of the genes involved in the biosynthesis of the precursors partially or totally incapable of coding for the natural enzyme involved in the biosynthesis of at least one precursor.
The genes liable to be mutated are preferably the genes involved in the biosynthesis of the precursor 4-dimethylamino-L-phenylalanine (DMPAPA). These are preferably the genes papA, papM, papB (SEQ ID No. 3) and papC (SEQ ID No. 2). The genes papA and papM have already been described in patent application PCT/FR93/0923.
By complementing the culture medium of mutant strains according to the invention with at least one original precursor, it turns out to be possible to orient the biosynthesis towards the novel streptogramins of general formula (I).
The precursors used in the context of the present invention are phenylalanine derivatives as well as a-ketocarboxylic acid derivatives which may be represented by the general formula: 
in which R and Rxe2x80x2 are defined as above in 2) and R10 is a hydrogen atom and R11 is an amino radical, or alternatively R10 and R11 together form an oxo radical.
As precursors which are suitable for the invention, mention may be made in particular of the following:
4-diethylaminophenylalanine, 4-ethylaminophenylalanine,
4-methylthiophenylalanine, 4-methylphenylalanine,
4-methoxyphenylalanine, 4-trifluoromethoxyphenyl-alanine,
4-chlorophenylalanine, 4-bromophenylalanine,
4-iodophenylalanine, 4-trifluoromethylphenylalanine,
4-tert-butylphenylalanine, 4-isopropylphenylalanine,
3-methylaminophenylalanine, 3-methoxyphenylalanine,
3-methylthiophenylalanine, 3-fluorophenylalanine,
4-tert-butylphenylpyruvic acid, 4-fluorophenylalanine,
3-ethoxyphenylalanine, 3,4-dimethylphenylalanine,
3-methylphenylalanine, 4-butylphenylalanine,
3-trifluoromethylphenylalanine and 3-ethylamino-phenylalanine, 4-aminomethylphenylalanine.
The preparation and separation of the components of the natural streptogramins of groups A [streptogramins of general formula (II)] and B [and in particular the products of general formula (VII)] are carried out by fermentation and isolation of the constituents from the fermentation broth according to or by analogy with the method described by J. Preud""homme et al., Bull. Soc. Chim. Fr., vol. 2, 585 (1968) in Antibiot. and Chemother., 5, 632 (1955) or 7, 606 (1957), in Chromatog. Sym., 2xc2x0 Bruxelles, 181 (1962), in Antibiot. Ann., 728 784 (1954-55), in U.S. Pat. No. 3,299,047 or in Streptogramine als Modelsysteme fxc3xcr den Kationentransport durch Membranen, Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Facultxc3xa4t der Georg-August Universitxc3xa4t zu Gxc3x6ttingen, Gxc3x6ttingen 1979, or as described below in the examples. In particular, in the case of pristinamycins, the components of the A and B groups are separated by suspending the crude streptogramin in an organic solvent such as an acetate (for example ethyl acetate), followed by filtration or centrifugation of the crude component of the A group and extraction of the component of the B group in aqueous acidic medium, followed by a re-extraction in chloromethylenic medium. The components of the A and B groups may also be separated by acidic extraction of a solution of crude streptogramin in methyl isobutyl ketone, followed by isolation by extraction of the component of the B group from the aqueous phase and isolation of the component of the A group by precipitation from the organic phase.
After separation, the components of the streptogramin B group may be purified by crystallization from an alcohol such as ethanol, methanol or isopropanol, from an acetate (for example isopropyl acetate or butyl acetate), from a ketone (for example methyl ethyl ketone) or from acetonitrile, or by chromatography. The components of the A group of general formula (II) may be purified by chromatography, eluting with an acetonitrile/water mixture.
Alternatively, the natural components of the A and B groups respectively of general formula (II) and in particular of general formula (VII) are prepared as described in French patent application 2,689,518, by separate fermentation.
Thus, according to the invention, it is now possible to obtain a novel purified and crystallized form of streptogramin in which the level of impurities, the definition and the constancy of the composition are improved and which moreover possesses improved in vivo activity and low toxicity. The novel co-crystallized combination will thus be able to overcome the absence of treatment by an antibacterial agent of this class in many countries.
The combinations according to the invention exhibit an in vivo biological action which is superior to that of the streptogramin combinations known hitherto, in the form of a mixture of powders.
The novel co-crystallized combination of a component of the streptogramin B group of general formula (I) and a component of the streptogramin A i group of general formula (II) is of particularly advantageous in vivo activity, in particular on Gram-positive microorganisms. In vivo, in mice, it proved to be active on Staphylococcus aureus IP 8203 at doses of from 25 to 50 mg/kg via the oral route.
Furthermore, the novel combination has no toxicity: no sign of toxicity is exhibited in mice at a dose of 150 mg/kg via the oral route (2 administrations).
The co-crystallized combination according to the invention may also be used as a means of purifying a minor component of streptogramins corresponding to the general formula (II).
Indeed, it has never been possible to purify a component of the A group of general formula (II) by crystallization.
It has now been shown that the component of the A group of general formula (II) can be-obtained in the pure state via the intermediate co-crystallized combination defined above.
According to the invention, when the co-crystallized combination is used as a means of purifying the component of general formula (II), the latter may be obtained by acidic extraction of a solution of the co-crystallized compound in a ketone (for example methyl isobutyl ketone), followed by isolation of the component of the A group by precipitation from the organic phase, when R or Rxe2x80x2 represents alkylamino or dialkylamino; or alternatively by high performance liquid chromatography.
Of particular interest are co-crystallized combinations which comprise at least one component of the streptogramin B group defined by the general formula (I), in which the symbols R1 and R2 are defined as above, and the symbol R3 represents a substituted benzyl radical of general formula (III) in which
1) on condition that R2 is simultaneously hydrogen, R represents NR4R5 for which one of the symbols R4 and R5 is a hydrogen atom or a methyl radical and the other is a methyl radical and Rxe2x80x2 is a chlorine or bromine atom, or represents an alkenyl radical containing 3 to 5 carbon atoms if R4 and R5 are methyl radicals, or alternatively
2) R is a hydrogen atom and Rxe2x80x2 represents an alkylamino or dialkylamino radical, an ether oxide residue or an alkylthio radical, or alternatively
R is an alkylamino radical containing 2 to 4 carbon atoms in a straight or branched chain, a dialkylamino radical in which the alkyl parts are identical or different and contain 2 to 4 carbon atoms in a straight or branched chain or a methylethylamino radical, a pyrrolidino radical, an alkenylalkylamino radical in which the alkenyl part contains 3 or 4 carbon atoms and the alkyl part is defined as above, an ether oxide residue, an alkylthio radical, an alkyl radical containing 1 to 6 carbon atoms in a straight or branched chain or a trihalomethyl radical, and Rxe2x80x2 is a hydrogen atom, or alternatively
R is an alkyl radical containing 1 to 6 carbon atoms in a straight or branched chain and Rxe2x80x2 represents a halogen atom, co-crystallized with at least one xe2x80x9cminorxe2x80x9d component of the streptogramin A group as defined above, it being understood that, except where specially mentioned, the alkyl radicals are straight or branched and contain 1 to 4 carbon atoms.
Among these products, the preferred combinations are co-crystallized combinations which comprise at least one component of the streptogramin B group defined by the general formula (I), in which the symbol R1 is an ethyl radical, the symbol R2 is a hydrogen atom, and the symbol R3 represents a substituted benzyl radical of general formula (III) in which:
1) on condition that R2 is simultaneously hydrogen, R represents NR4R5 for which one of the symbols R4 and R5 is a hydrogen atom or a methyl radical and the other is a methyl radical and Rxe2x80x2 is a chlorine or bromine atom, or represents an allyl radical if R4 and R5 are methyl radicals, or alternatively 2) R is a hydrogen atom and Rxe2x80x2 represents an alkylamino or dialkylamino radical, a methoxy, ethoxy, allyloxy or trifluoromethoxy radical or an alkylthio radical, or alternatively
R is an alkylamino radical containing 2 to 4 carbon atoms in a straight or branched chain, a dialkylamino radical in which the alkyl parts are identical or different and contain 2 to 4 carbon atoms in a straight or branched chain or a methylethylamino radical, a pyrrolidino radical, an allylalkylamino radical in which the alkyl part is defined as above, a methoxy, ethoxy, allyloxy or trifluoromethoxy radical, an alkylthio radical, an alkyl radical containing 1 to 6 carbon atoms in a straight or branched chain or a trifluoromethyl radical, and Rxe2x80x2 is a hydrogen atom, or alternatively
R is an alkyl radical containing 1 to 6 carbon atoms in a straight or branched chain and Rxe2x80x2 represents a halogen atom, co-crystallized with at least one xe2x80x9cminorxe2x80x9d component of the streptogramin A group as defined above, it being understood that, except where specially mentioned, the alkyl radicals are straight or branched and contain 1 to 4 carbon atoms.